Monoclonal antibodies are utilised widely in both the academic and commercial pharmaceutical and biotechnology sectors. They are used to detect specific entities, commonly referred to as antigens, such as—but not exclusively—proteins or peptides, through an interaction with a specific site on that entity known as the epitope. The same epitope may be found on different proteins, similarly any one protein may display several different epitopes. Such reagents are commonly used in basic research, diagnostic, assay development and more recently therapeutic applications.
Antibody specificity is defined as the degree to which an antibody will recognise and distinguish one epitope (or subset of epitopes) from a diverse repertoire of other epitopes. Although the absolute degree of specificity required of the antibody may be application specific, it is generally the case that the investigator desires maximum specificity from the antibody, thus minimising the likelihood of obtaining false results as a consequence of non-specific interactions. The greater the requirement for specificity, the more challenging it can become to generate such a reagent. Thus it follows that improvements in the efficiency of producing, selecting and cloning cells which secrete monoclonal antibodies will increase the likelihood of being able to obtain a reagent with the required application-specific characteristics.
Although well established and widely utilised, the fundamental method for producing antibodies by fusing splenic lymphocytes with myeloma cells has remained largely unmodified for the past 25 years. Due to some of the limitations inherent in this technique, many new technologies have been developed to produce protein (antigen/epitope) recognition reagents with similar characteristics—and in some cases structures—to the traditional monoclonal antibody. These include phage display, phagemid display, scFV and others (Adams and Schier 1999; Walter et al. 2001). Despite the potential advantages and promise of many of these new technologies, antibodies produced by the hybridoma method—or a modification thereof—developed by Kohler and Milstein (1975) remains the method of choice in most in-house and contract laboratories. Thus an advance which significantly improves the efficiency, reliability and ultimately the likelihood of engineering a useful reagent via this method—as described in this invention—provides a significant breakthrough.
Current Methods of Producing Monoclonal Antibodies and their Limitations
Activation of β-lymphocytes can lead to the production of an antibody specific for a given antigen. However the utility of these β-lymphocytes is limited for in vitro antibody reagent production as such cells are short-lived in cell culture (Kohler and Milstein 1975). Thus methods have been developed to enable these antibody secreting cells to survive in culture by fusing them with immortalised cell lines (myelomas), which themselves can grow indefinitely in culture. Many of the myeloma lines used today were originally antibody-secreting themselves, such as Sp2 cells. Thus the challenge faced by the pioneers of monoclonal antibody technology was threefold: 1) to develop appropriate immortalised cell lines which themselves secrete no antibodies but would enable immortalised growth of a myeloma/β-lymphocyte hybridoma, 2) to optimise the fusion of the β-lymphocyte and myeloma line, and 3) to devise a method of selecting hybridoma cells over both non-fused myeloma and β-lymphocyte cells. Kohler and Milstein in (1975) were the first to describe a method which achieved these goals by fusing the β-lymphocyte with a myeloma line using Sendai virus. Later it was discovered that polyethylene glycol (PEG) could achieve the same aim. PEG is widely used today as the fusion agent of choice; the reagent often referred to as the ‘fusogen’.
The host used for monoclonal antibody production is in most cases a Balb/c or Balb/c hybrid mouse, since most of the myeloma cell lines are derived from Balb/c mice. The antigen may be a peptide, pure protein, partially purified protein or perhaps a non-purified tissue sample. It may also be soluble or insoluble. The immunisation schedule often involves the co-injection of an adjuvant such as freunds to assist in the induction of an immune response. Additionally less immunogenic antigens and peptides are often coupled to a carrier such as key-hole lympet haemocyanin (KLH) to increase their immunogenicity. The immunogenicity defining the likelihood that the antigen will induce the required immune response. The progress of the immune response is measured by taking serum samples throughout the schedule and testing—by methods such as enzyme linked immunoabsorbent assays (ELISA) or western blotting—for the presence of antibodies specific for the antigen of interest. Once a strong immune response against the antigen of interest has been detected the, β-lymphocytes (usually from spleen) are harvested and fused with the myelomas. The precise timing of the spleen harvest with respect to the final immunisation, can influence the efficiency of the fusion (Stahl et aL 1983; Cianfriglia et al. 1986; Cianfriglia et al. 1987).
The Fusion
The success of the fusion between the spleen derived β-lymphocytes and the myeloma cell line may be controlled—at least to some extent—by a number of factors. These include, although not exclusively; the fusogen, temperature, cell mixing protocol, ratio of spleen to myeloma cells, time of fusion, cell fusion recovery protocol, media/buffer batches and of course the investigator. The precise details of the protocol and success of the fusion often vary from laboratory to laboratory and even from experiment to experiment (Igarashi and Bando, 1990).
The fusion is catalysed by the addition of polyethylene glycol (PEG) to a suspension of spleen and myeloma cells. The success of the procedure is very much dependent on the skill and experience of the operator, as the freshly fused cells are sensitive to mechanical and chemical disruption. The method of PEG addition to the cells and the formulation of PEG used has been optimised to achieve fusion frequency of around 6×10−6 to 3×10−5 (Lane et al. 1984; Lane 1985; Lane et al. 1986). The fusion frequency being defined as the number of hybridomas generated divided by the number of lymphocyte cells used in the fusion (approximately 1×108 cells total from a single spleen).
An alternative method to the use of PEG as the fusogen is ‘electrofusion’, where an electrical field is used to fuse the spleen cells with myelomas. Under optimal conditions it has been possible to increase the fusion frequency to 10−3-10−4 (Schmitt et al. 1989) or 10−3 (van Duijn et al. 1989), around an 80-fold improvement on the traditional PEG method described above. The number of antigen-specific hybridomas as well as the total number of hybridomas was similarly increased by this method, the improvement being independent of the immunisation procedure, the antigen or the source of the lymphocytes. Hui et al. (1993) has analysed a number of publications where the PEG and electrofusion methods have been compared directly in the same experiment. They conclude that on the whole, the electrofusion procedure offers an improvement in the fusion rate, either measured by the number of HAT resistant or antibody producing clones generated. A further report indicates approximately a 10-fold improvement in fusion rates—utilising the electrofusion technique compared to the PEG method—across thirty-six experiments (Karsten et al. 1993). However the rate of success is not consistent across all laboratories, thus demonstrating that there is still scope for refinement in the methodology and it's reproducibility.
Despite an increase in the fusion rate achieved by methods such as those described above, a percentage of hybridomas secrete no antibody at all. This is not thought to be due to fusion of myelomas with T cells, as there is no evidence for the presence of myeloma×T-cell heterokaryons from viable hybrids (Kohler et al. 1977; Clark and Milstein 1981). Problems may be encountered after the plating of fused cells due to overgrowth of hybrid cells by macrophages, fibroblasts and P cells (van Mourik et aL 1984). One approach to help overcome these issues is to enrich for immunoglobulin secreting spleen cells by methods such as density centrifugation (van Mourik and Zeijlemaker, 1986) before fusing with the myeloma cells.
By bringing the spleen cells displaying cell surface receptors to the antigen (used to immunise the mouse) in close proximity to the myeloma cell, the efficiency of the fusion can be increased still further without the need for the density separation step. This procedure improves the efficiency of the fusion in two ways. Firstly by reducing the relative number of unwanted fusions, that is fusions between myeloma cells and spleen derived cells that are not capable of secreting antibodies. Secondly by selectively fusing antigen secreting cells that present only cell-surface receptors that bind the antigen of interest (Lo et al. 1984; Tsong and Tomita, 1993). This has been achieved by creating a ‘bridge’ between these two cells taking advantage of the strong interaction between biotin and streptavidin (Yuan et al. 2000). Thus an avidin-antigen conjugate is generated which binds the cell-surface antibody receptors on the splenic lymphocytes. Biotinylation of the myeloma cell brings it into close proximity with the antigen recognising lymphocyte population through a strong interaction with the avidin-antigen conjugate. Thus increasing the chances of successfully fusing lymphocytes of interest with the immortalising myeloma cell line by PEG fusion (Reason et al. 1987).
An alternative method to the use of PEG for catalysing the fusion of bridged spleen with myeloma cells is the application of the electrofusion technique (Lo et al. 1984; Hewish and Werkmeister 1989; Conrad and Lo 1990; Werkmeister et al. 1991). A combination of both methods yielding fusion frequencies of as high as 10−2, or a 10-fold improved (Tomita and Tsong 1990) over the electrofusion method alone (van Duijn et al. 1989).
A further approach to increasing the fusion rate through bringing the cells into closer contact utilises Neuramidase to remove sialic acid from the cell membrane (Igarashi and Bando 1990). It is thought that the extracellular membranous sialic acid prevents close intercellular contact and hence the likelihood of PEG facilitated fusion. Indeed such treatment yielded approximately twice as many HAT resistant clones and eight-fold more antigen-specific clones over non-treated cells. Combinations of the above techniques may improve further the reliability and efficiency of the cell-fusion procedure. Other methods shown to increase the fusion efficiency by 10- to 50-fold include the adoptive transfer of spleen cells from immunised animals to X-irradiated syngenic recipients, followed by antigen boosting and culturing of spleen cells with the antigen prior to the fusion (Siraganian et al. 1983).
In addition to the low frequency of fusion that is achievable—even utilising many of the modified fusion techniques described above—there are other inherent limitations to this approach of immortalising β-lymphocytes. The fusion methods described above do not discriminate between the cell-cycle phase of either of the fusion partners. Thus fusions may occur between cells in mitosis or interphase. This can lead to a phenomenon known as premature chromosome condensation, or PCC (Westerwoudt, 1985) which, under some circumstances, can lead to the disruption of cell division.
A further factor that can influence the final success of the fusion is chromosome stability (Westerwoudt, 1986). Thus after fusion the new cell contains a full complement of chromosomes from both parent cells. This must be reduced to a normal complement of chromosomes for sustained growth. This inevitably requires a loss of the equivalent of one complement of chromosomes, which may include chromosomes harbouring the antibody expressing genes. It is estimated that approximately 50% of hybridomas that initially express antibody after successfully fusing, loose their ability to do so due to chromosome loss (Clark and Milstein 1981). Additionally hybridomas may not survive in the HAT selection media due to loss of the spleen derived HPRT gene, which is located on the X-chromosome (Taggart and Samloff 1982).
Even if the cell is able to successfully fuse and adopt a hybrid karyotype of chromosomes, hybridoma lines often display some degree of karyotipic instability. This is often the reason why seemingly stable hybridoma lines suddenly stop secreting antibodies. Thus the long-term karyotipc stability of any hybridoma line is an inherent characteristic of each hybrid and is a function—at least in part—of the process of integration of the two parental genomes (Westerwoudt, 1986). Freeze-thawing of cells has been used successfully to determine which lines are unstable, since this process results in the loss of antibody production in unstable lines (Pravtcheva and Ruddle, 1983).
Selection of Hybridomas
The selection of hybridomas utilises specific growth features of both the spleen and myeloma cells. Firstly, as discussed above, spleen cells are unable to grow in culture, thus unfused cells die. Myeloma cells defective in the enzyme hypoxanthinephosphoribosyltransferase (HPRT) were originally selected by Kohler and Milstein (1975) since they are unable to grow in medium containing hypoxanthine, aminopterin and thymidine (HAT medium). Since the aminopterin blocks the main pathway for DNA synthesis while the rescue pathway requires HPRT to utilise the exogenous hypoxanthine and thymidine. Thus myeloma cells die in HAT medium while the spleen cells provide the HPRT gene that enables growth of the hybridomas.
Although some improvements have been made to the precise experimental details, the fundamental method for immortalising β-lymphocytes through fusion with myelomas has remained unchanged since the description of the technique by Kohler and Milstein in 1975. One aspect of this invention presents a method that increases the efficiency of immortalisation to a theoretical 100%, while greatly simplifying the whole procedure; eliminating the fusion step through the use of inducible oncogene(s)/antisense expression or post-translational activation of oncogenes through the use of transgenic technologies.